New approaches for developing viral
vaccines against influenza and
respiratory syncytial virus
December 8, 2015
Virology-2015 Atlanta
Sang-Moo Kang
Georgia State University
1918 (H1N1) : The worst and greatest pandemic
- deaths of 40 to 100 million people worldwide.
Lung inflammation
3-500,000
deaths
rate per 100,000 p-years
The yearly burden of influenza
deaths
influenza deaths
125
100
75
50
25
0
<1
5-49
50-64
65+
Age (years)
hospitalizations
influenza hospitalisations
rate per 10,000
40
3-5 million cases
of severe illness
1-4
30
20
10
0
0-4
5-9
10-19 20-34 35-44 45-54 55-64 65+
Age (years)
cases
influenza cases
1 billion cases of
influenza
rate per 100,000
100
75
50
25
0
0-4
5-9 10-19 20-34 35-44 45-54 55-64 65+
Age (years)
90% of deaths occur in the elderly (>65 years)
MMWR, Aug 2008
(From Yuna Lee)
Influenza virus
Swine new
H1N1
Swine
2009
H1N1
H7N9
H7N9
2013-
379 deaths/
638 cases
Over 18,000
deaths
127 deaths/
419 cases)
(From Eunju Ko)
Challenges in influenza vaccination
1. No licensed vaccines against avian influenza viruses.
2. No good cross protection against drift epidemic and new
pandemic flu.
3. Approximately 6 months’ time for vaccine production using
chicken eggs.
4. Vaccine delivery (cold chain, syringe-needle flu shots,
medically trained persons)…. Microneedle vaccine delivery.
5. Continuous mutations in natural reservoirs (humans, wild
birds, poultry, pigs, etc..).
Structural similarity of Virus and VLPs
Non-replicating VLPs
(Virus-like particles )
as a new vaccine modality
Replicating Virus
M1
M2
HA
M1
NA
HA
NA
Viral
Genomes
In virions
Gag
Env
Production of Avian influenza H5 VLPs in insect cells
A
Recombinant
Baculovirus
HA1
HA2
WT H5 RRRKKR
-TR
Mutant H5
Influenza
VLPs
B
1
Anti-HA
2
3
M1
4
C
kDa
VLP
HA0
75
HA1
50
37
25
D
1
rHA
2
3
4
75
75
50
37
50
37
Trypsin (-)
Trypsin (+)
1
3
2
4
(Song et al., 2010. Virology)
Pandemic potential influenza H5N1 and
pandemic 2009 H1N1 VLP vaccines
are immunogenic and protective (1)
1. Avian H5 and pandemic 2009 H1 VLPs are highly immunogenic,
inducing virus-specific antibody responses.
2. Avian H5 and pandemic 2009 H1 VLPs induce IgG2a, IgG2b
antibodies as major isotypes and IFN-gamma cytokine secreting cells.
3. A single intramuscular immunization induces protective immunity and
long-lasting plasma and memory B cells
4. VLP vaccines are superior to soluble protein and split vaccines in
inducing protective immunity in mice and ferrets.
Clinical Trials of virus-like particle (VLP)
influenza vaccines
1. Novavax Trivalent seasonal influenza VLP vaccine (2008-2009):
second phase clinical phase II
2. Novavax 2009 H1N1 pandemic influenza VLP vaccine (2011): phase
clinical phase II
3. Novavax pandemic potential Avian H5N1 VLP vaccine (2011) : FDAapproved phase I/II human clinical study.
4. Novavax pandemic potential Avian H7N9 VLP vaccine (2013) : phase
I/II human clinical study, Saponin-based ISCOMATRIX adjuvant.
How is it possible to overcome the strainspecific protection of influenza vaccination?
HA is abundant, larger, dominates immune responses
HA
NA
Virus surface
proteins
M2
HA VLPs
NA VLPs
Surfaces of VLPs
M2 VLPs
Designing better vaccines by molecular engineering?
(2nd generation M2e5x VLP)
M2
Wild type M2
Human
TM
C-tail
Tandem repeat M2e5x
M1
Human
Human
Swine
Avian I
Avian II
HA-TM-tail
M1
M
1
2
3
4
5
6
50kD
37kD
25kD
20kD
15kD
1.
2.
3.
4.
5.
6.
Influenza virus, 10 ug
Influenza virus, 5 ug
Influenza virus, 1 ug
M2e5x VLP, 0.1 ug
M2 WT VLP, 1 ug
No M2
(Kim MC et al., 2013 Mol Therapy)
M2e5x VLP induce higher levels of diverse M2e antibodies
than virus
M2e5x VLP
A/PR8 H1N1 virus
A/Phil H3N2 virus
A/CA/2009 H1N1 virus
Homo.M2e4x VLP
Human M2e
Human type ELISA
3.0
5xM2e VLP boosted
PR8 infected
H3N2(Phil) infected
2009 H1N1 infected
Naive sera
Li 4xM2e VLP vaccinated
OD(450nm)
2.5
2.0
1.5
1.0
0.5
VLP vaccination
Virus infection
0.0
0
2
3
4
5
6
Avian M2e
Swine M2e
Major avi type ELISA
Swine type ELISA
3.0
5xM2e VLP boosted
PR8 infected
H3N2(Phil) infected
2009 H1N1 infected
Negative
Li 4xM2e VLP vaccinated
2.5
2.0
1.5
1.0
5xM2e VLP boosted
PR8 infected
H3N2(Phil) infected
2009 H1N1 infected
Naive
Li 4xM2e VLP vaccinated
2.5
OD(450nm)
3.0
OD(450nm)
1
Serum dilution(50, 200, 800, 3200, 12800, 51200)
2.0
1.5
1.0
0.5
0.5
0.0
0.0
0
1
2
3
4
5
6
Serum dilution(50, 200, 800, 3200, 12800, 51200)
0
1
2
3
4
5
6
Serum dilution(50, 200, 800, 3200, 12800, 51200)
(Kim MC et al., 2013 Antiviral Res)
M2e5x VLPs confer better protection against
Human H3N2 and avian H5N1
M2e5x VLP or
M2WT VLP
(10 ug)
Intramuscular
Weeks 0 and 4
Lethal challenge
Avian H5N1 (rg A/Vietman/1203/04)
Human H3N2
(A/Phil/82)
110
M2e5x
M2WT
Naive
Body Weight(%)
105
100
95
90
85
80
75
M2e5x
M2WT
Naive
105
Body Weight(%)
110
Figure 4
100
95
90
85
80
75
70
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Days post infection
70
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Days post infection
Kim MC et al (Mol. Therapy, 2013)
Hypothesis:
Supplementing human vaccines with M2e5x VLP
will improve the cross protective efficacy?
Commercial
Human Split vaccine
(Green Cross)
M2e5x
VLP
Supplementing human vaccines with M2e5x VLP
confers improved cross protection compared to the vaccine only
rgH5N1 virus challenge
110
Split+5xM2e
Split Vac
Naive
H5N1 virus
(re.A/VN/1203)
M2e5x
VLP
Body Weight(%)
105
Split
human
vaccine
100
95
90
85
80
75
70
H3N2 virus
(A/Phil/1203)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Days post infection
A/Phil (H3N2) challenge
Split+5xM2e
Split Vac
Naive
Body Weight(%)
105
100
95
90
85
80
Survival rate (%)
100
110
Split+5xM2e
Split Vac
Naive
75
50
25
75
0
70
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Days post infection
Days post infection
(Kim et al., 2014, Mol Ther)
Recombinant Influenza Virus Carrying M2e4x in
a chimeric hemagglutinin conjugate induces
cross protective antibody responses
A. N-terminal chimeric 4xM2e-HA (N)
SP
N
HA1-HA2
M2eH-M2eH-M2eS-M2eA
4xM2e-HA
B)
A)
100 nm
100 nm
Protective efficacy to
heterosubtypic influenza A viruses
Body Weight (%)
105
A/California (H1N1)
P<0.05
P<0.01
105
A/Philippines (H3N2)
P<0.05
P<0.01
105
A/Mandarin duck (avian rgH5N1)
P<0.05
105
100
100
100
100
95
95
95
95
90
90
90
90
85
85
85
85
80
80
80
80
75
75
75
A/Vietnam (rgH5N1)
P<0.05
rg/M2e4x-HA
wt/HA
Naive
75
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Days post infection
Days post infection
Days post infection
Days post infection
Summary (2)
Experimental flu universal vaccine
New M2e5x VLP vaccines can confer broad heterosubtypic cross
protection in pre-clinical animal models (mice, ferrets)
Supplementation with M2e5x VLP significantly improves the cross
protective efficacy of current flu vaccines
M2e antibodies, CD4 & CD8 T cells, dendritic/macrophage cells, Fc
receptors are important for M2e-immune mediated protection.
Preclinical Efficacy of
experimental vaccines against
respiratory syncytial virus
Respiratory Syncytial Virus (RSV)
• 64 – ~120 million hospitalizations and 160,000 – 234,000 deaths globally (up to
•
•
•
•
•
940,000 RSV pneumonia associated deaths mostly in developing countries:
Luksic, 2013; Smith, 2013; Shi 2014)
Bronchiolitis and pneumonia in children under 1 (or 5) years old children
Recurrent wheezing and asthma
immunocompromised patients and infants born prematurely severe respiratory
illness requiring hospitalizations
no vaccine exists
Palivizumab, a monoclonal antibody directed against RSV surface fusion protein
Clinical Trials of Novavax RSV F nanoparticle protein vaccines
Novavax RSV F nano-particle (30 – 40 nm) protein vaccines
1. Phase I clinical study 18-19 years of age (2013)
2. Phase II clinical study (350 healthy women of child bearing ages
18 – 35 years of ages (2015)
3. Planning phase III study (2016) : Maternal immunization.
Production and characterization of RSV F and G VLPs in insect cells
Recombinant
Baculovirus
RSV-G Virus like particles
RSV-F Virus like particles
50 nm
RSV-F Virus like particles
KD
20ug
5ug
50 nm
RSV-G Virus like particles
KD 20ug
1ug
70
F
75
25
M1
25
5ug
1ug
G
M1
(Quan et al., 2011, J. Inf. Dis.)
F or G VLP controls lung viral clearance similar to FI-RSV (and live RSV)
Lung RSV titers
400
200
SV
Li
ve
-R
-V
G
ai
ve
N
LP
0
FF
SV
-G
600
R
R
ai
v
N
SV
-F
10000
800
f.
20000
Naive-Inf.
FI-RSV
FFG-VLP
Live-RSV
1000
-In
30000
e
Lung virus titer
( PFU/mouse)
40000
Lung virus titer (PFU/mouse)
Lung RSV titers
SV
VLP vaccines
(RSV F + G )
Live RSV
challenge
-R
Intramuscular
immunization
FI
FI-RSV (IM)
Live RSV (IN)
RSV-F or G VLP immunization
(Quan et al., 2011, J. Inf. Dis.)
(Ko et al., 2014, Nanomedicine)
The most challenging difficulty in
developing RSV vaccines:
Vaccine safety?
A safe vaccine should not induce “Vaccineenhanced pulmonary (lung) respiratory
disease?
SV
LP
***
3
2
1
0
Inflamation Scores of
Interstitial Spaces
***
N
iv
e
in
fe
ct
io
n
FI
-R
SV
FF
G
-V
LP
Li
ve
-R
SV
-R
-V
SV
C
iv
e
ve
G
-R
FI-RSV
(Hwang et al., 2014, Anti. Viral Res)
N
Li
FF
n
Naïve-infection
FI
4
io
e
0
fe
ct
1
Inflamation Scores of
Blood Vessels
2
iv
SV
LP
***
N
-R
-V
SV
B
in
ve
G
-R
3
Li
FF
e
n
iv
io
N
fe
ct
FI
in
4
e
e
Naïve
iv
iv
H&E
A
N
N
Inflamation Scores of
Airways
RSV VLP vaccines do not cause pulmonary
inflammatory disease upon live RSV challenge (1)
FFG-VLP
Live-RSV
4
D
3
***
*
2
1
0
l
d
RSV VLP vaccines do not cause inflammatory
eosinophilia upon live RSV challenge (2)
Naïve
Naïve-infection
FI-RSV
Live-RSV
f
e
r
v
PAS
B
h
80
60
S V
L i
v e
-R
L P
-V
F F
G
N
a i
v e
0
S V
o
40
F I
-R
n
i
***
20
E
S V
L i
v e
-R
L P
-V
S V
F I
-R
F F
G
N
N
0
a i
v e
%
20
a i
v e
-I
n f
.
s
P
40
***
100
a i
v e
-I
n f
.
o
p
***
N
***
60
A
S
P
i
l
o
s
s
p
i
t
e
i
A
H&CR
i
e
FFG-VLP
(Hwang et al., 2014, Anti. Viral Res)
Cotton rats are a more relevant animal
model for RSV vaccine studies
RSV F
FI-RSV
Live RSV
Naive
Intramuscular
immunization
Live RSV
challenge
Summary
• RSV VLP vaccines induce protection without vaccine-enhanced
disease in mice and cotton rat animal models.
•
•
•
•
RSV F specific IgG2a dominant production
Neutralizing RSV activity and virus clearance
T helper 1 immune responses
No eosinophilia and inflammation in the lungs of mice and cotton rats
• FI-RSV caused severe vaccine-enhanced disease (clinical trials,
various animal models)
•
•
•
High immunogenic (RSV specific antibody production)
T helper 2 immune responses
Severe eosinophilia and inflammation in lung after RSV challenge
• Live RSV does not provide long-term immunity, recurring
infections
•
•
Good immune responses and protection
Short memory duration and reinfection throughout life
(Hall CB et al., J Infec Dis., 1991)
Acknowledgements
Research support
NIH/NIAID
GSU RF
Georgia State University
Yuna Lee
Kihye Kim
YoungMan Kwon
Youngtae Lee
Minchul Kim
Eunju Ko
Hyesuk Hwang
Yujin Jung
Youri Lee
Yujin Kim
Ye Wang
Collaborators
(Former members)
Emory University
Martin Moore
CDC (Atlanta, USA)
Ruben Donis
Ian York
Nedzad Music
Mercer University
Martin D’Souza
Georgia Inst. Technology
Mark Prausnitz
Eunju O
Sieun Yoo
Jae-Min Song (Sungshin U)
Fu Shi Quan (KyungHee U)
JongSeok Lee
Minkyoung Cho
Vu NGO
Daegoon Yoo (UGA)
Minkyung Park (C. W. U)
Yeu-Chun Kim (KAIST)
Ioanna Skountzou (Emory)
Sailaja Gangardhara (Emory)
BEAMS BIOTECH
Cheol Kim
Jongsang Lee
QIA
Younjeong Lee
Research materials
University of Alberta
Hyo-Jick Choi
Carlo Montemagno
Green Cross
(split vaccine)
NIH BEI
Thank you
Questions?